This invention is related to the production and use of novel monoclonal antibodies reactive with any part of endotoxin core of Gram-negative bacteria, and self-reproducing carrier cells containing genes which code for monoclonal antibodies reactive with endotoxin core. The invention is directed to the antibodies, to processes of preparing the antibodies, to diagnostic, prophylactic, and therapeutic methods and compositions employing the antibodies, and to investigational, pharmaceutical, and other methods and compositions employing the antibodies.
Gram-negative bacteria are a ubiquitous and diverse group of microorganisms that cause a number of serious and often life-threatening infections. Lipopolysaccharides are the principal biochemical constituents of the external covering or cell wall which characterizes all Gram-negative bacteria. These lipopolysaccharides, or endotoxins as they are called, play a major pathophysiologic and immunologic role in Gram-negative infections. Antibodies directed toward bacterial lipopolysaccharides constitute a critical host defense against these toxic substances and against the organisms which produce them.
In addition to their direct role in Gram-negative infections, endotoxins possess diverse and potent biological activities which, when coupled with the widespread presence of endotoxins in man's internal and external environments, endow them with great medical, scientific, and economic importance.
In general, bacterial lipopolysaccharides consist of a highly variable outer region composed of repeating oligosaccharide (sugar) units comprising the so-called "0-specific side-chain", and a relatively constant core region containing a limited number of sugars, often including the trisaccharide 2-keto-3-deoxyoctonate (KDO), and the biologically active lipid A moiety. Lipid A's derived from a number of distinct bacterial groups show a close structural relationship, and in most cases studied, lipid A consists of a .beta.1,6-linked glucosamine disaccharide with ester- and/or amide-linked long chain fatty acids and with other possible substitutions. The core region, which is sometimes referred to as core glycolipid or endotoxin core, may be considered a lipopolysaccharide (or endotoxin) of Gram-negative bacteria that is lacking its 0-specific side-chain. In addition, the endotoxin core structure may itself be incomplete on the basis of missing core sugars and/or other substituents. Thus, endotoxin core is incomplete lipopolysaccharide, lacking part or all of the 0-specific side-chain, and, in some cases, also lacking core sugars and/or other substituents while usually retaining lipid A. (See E. Th. Rietsche et al., Scandinavian Journal of Infectious Diseases, Supplement 31:8-21, 1982, which is hereby incorporated by reference).
Significantly, most Gram-negative bacteria, representing diverse genera and species and including almost all which are pathogenic for man, share highly analogous, immunologically cross-reactive endotoxin core structures. For purposes of this specification, endotoxin core will be defined as that part of the lipopolysaccharide or endotoxin of Gram-negative bacteria comprised of complete or incomplete, substituted or unsubstituted lipid A covalently bound to substituted and/or unsubstituted core sugars, said lipid A characterized, for example, by a phosphorylated .beta.1,6-linked glucosamine disaccharide with ester- and/or amide-linked long chain fatty acids, and said core sugars distinguished by their location in an interconnecting position between lipid A and the repeating oligosaccharide units of the 0-specific side chain in the intact lipopolysaccharide molecule.
Antibodies to 0-specific side chains in the variable outer region of lipopolysaccharides are species- and typespecific, with protective activity derived from their ability to promote the phagocytosis (engulfment) and killing of infecting organisms by host phagocytes (white blood cells). In contrast, antibodies directed toward the lipid A-containing inner core region are broadly cross-reactive among a wide variety of Gram-negative bacteria and are thought to act by neutralizing the biological activities of endotoxin.
Antibodies are produced by living cells called plasma cells which are specialized for that function. Plasma cells are derived from other cells called B lymphocytes which bear receptors on their cell membrane with the same antigen specificity as the antibodies synthesized and secreted by the plasma cells. Each immunocompetent B lymphocyte and its progeny (clone) bears receptors with unique specificity. Foreign substances (antigens) bind to receptors on B lymphocyte cell membranes and stimulate these specific B lymphocyte clones to proliferate, differentiate into plasma cells, and produce specific antibodies. In simplified terms, there are as many lymphocyte clones as there are specific antibodies, and as many specific antibodies as there are distinct antigens. Conversely, each specific antibody is produced by plasma cells derived from a single clone of immunocompetent lymphocytes, and an individual exposed to (i.e., immunized with) a specific antigen, produces specific antibody to that antigen through expansion of the appropriate lymphocyte clone.
Antibodies are characterized by exquisite specificity for the antigen toward which they are directed. In reality, most antigens contain more than one antigenic site, so that multiple antibodies may be directed at single antigens. In addition, different antibodies with varying affinities (strength of antigen binding) may be directed toward single antigenic sites. Since multiple antibodies directed toward the same antigen are derived from different lymphocyte clones, they are referred to as "polyclonal" antibodies. The normal antibody response to most antigens is polyclonal. At the same time, a single antibody may react with multiple antigens or antigenic sites which have common or analogous molecular structures; such an antibody is called a cross-reacting antibody.
It can thus be appreciated that the total antibody repertoire of an individual is enormous in respect to both size and breadth, and that natural exposure of an individual to an invading microorganism or immunization with a complex antigen will result in a polyclonal antibody response. The serum from such an individual will contain a complex admixture of pre-existing and new antibodies characterized by a multitude of specificities and affinities.
Traditionally, polyclonal antibodies have been prepared by immunizing an animal or man with the material (antigen) toward which antibodies are sought. If the goal of immunization is the prevention or treatment of a specific disease, intoxication or infection, an individual may be immunized directly with the appropriate antigen (active immunization), or administered pre-formed antibodies or immune serum prepared by prior immunization of another individual with the same antigen (passive immunization). Both types of immunization have major shortcomings. In the case of active immunization, there may be insufficient time to achieve an adequate antibody response to prevent or treat a particular infection or disease. In addition, it is sometimes impossible to accomplish active immunization because of ineffective vaccines, the inability of certain groups (e.g., immunosuppressed persons and infants) to respond to vaccination, or untoward reactions sometimes associated with active immunization. Passive immunization, on the other hand, lacks specificity and is associated with a significant risk of transmissible infections such as hepatitis or other adverse reactions. Antisera, or immunoglobulins prepared from antisera, contain not only the desired antibody, but literally thousands of other antibodies as well. In fact, the desired antibody usually represents only a small fraction of the total antibody present in such antisera. It may be difficult, therefore, to achieve adequate levels of this antibody through passive immunization using such antisera. This poses additional risks to the patient as the infusion of large volumes of antisera or immunoglobulin greatly increases the likelihood of serious adverse reactions and infusion-related infections.
The non-therapeutic uses of polyclonal antibodies, as for example in immunological research and various biotechnological applications, may also be seriously hampered by the variability of antibody responses to many antigens and the lack of specificity of antisera which contain a wide variety of antibodies.
Thus, the heterogeneity and diversity of naturally acquired or immunization-induced antibodies, and the unpredictability of antibody responses to antigenic stimuli, are factors which seriously limit the practical use of these polyclonal antibodies for clinical or scientific purposes.
While the foregoing discussion of the limitations of polyclonal antibodies has been stated in general terms, these same limitations apply to the therapeutic and non-therapeutic uses of polyclonal antibodies directed toward endotoxin core specifically.
In summary, Gram-negative bacteria are a widely prevalent group of microorganisms that commonly cause serious and often life-threatening infections. These bacteria all produce lipopolysaccharides or endotoxins which confer upon them important pathogenic and immunologic properties. Endotoxins have broad medical, scientific and economic significance on the basis of their varied biological properties that goes well beyond their role in Gram-negative infections. Endotoxins from a wide variety of sources share a common or highly analogous core structure. Antibodies to this core structure cross-react with lipopolysaccharides produced by many different Gram-negative bacteria. These cross-reactive antibodies neutralize the biological activities of endotoxin, and appear to provide protection against serious Gram-negative infections. The utilization of naturally acquired or immunization-induced polyclonal antibodies reactive with endotoxin core is limited by the low immunogenicity of endotoxin, the lack of specificity of polyclonal antiserum, and the adverse reactions associated with conventional active or passive immunization.